| Objective:As the rapid development of immunological and moleeular biologieal technologies in the Past 20 years,more and more people are interested in the role of tumor markers.At present,many markers play a decisive role in the treatment and screening,among which ER,PR,HER-2 and Ki-67 in the field of breast cancer are relatively clear and guide the work of clinicians.However,ER,PR,HER-2 and Ki-67 as specific diagnostic and predictive indicators of breast cancer,its specificity and sensitivity are not ideal.It is necessary to search for better molecular markers related to prognosis,so as to provide help for clinical understanding of biological behavior of breast cancer and guiding treatment.Receptor-associated protein 80(RAP80)is a member of BRCA1-A complex which regulate the cell cycle checkpoint and DNA damage repair in the nucleus1.RAP80 function is intimately linked to its ability to bind ubiquitin and Small ubiquitin-like modifier at DNA double-strand breaks.Inactivation of RAP80’s SUMO-interacting motif(SIM)or either of its two ubiquitin-interacting motifs(UIMs)impairs its localization to DSBs and increases sensitivity to ionizing radiatio.Given the importance of RAP80 in maintaining genomic integrity,much work over the past ten years has gone into understanding how RAP80 regulates DSB repair and uncovering the mechanism by which RAP80 interacts with ubiquitin and SUMO.In this review,We studied the expression of RAP 80 in breast cancer and its paired normal breast tissues,and analyzed its role in the biological behavior of breast cancer cells.Materials and Methods:Patients and specimensFresh breast tissue samples were collected from Department of Breast Surgery,the First Affiliated Hospital of China Medical University between Feb 2010 and Sep 2011,including the ductal carcinoma in situ,invasive breast ductal cancer tissues and its paired normal breast tissues(with>2cm distance from the primary cancer tissue).None of patients undergo chemotherapy,radiotherapy or adjuvant treatment before surgery.Patients’ ages ranged from 24 to 79,with an average age of 51.8 years old.Clinicopathological information was reviewed using the hospital medical records.All patients enrolled in this study have signed an informed consent form to agree to participate in this study and for publication of the results.The study protocol was reviewed and approved by the Ethics Committee of China Medical University(Shenyang,China)and of the participating hospital.Immunohistochemistry4-μm-thick formalin-fixed and paraffin-embedded specimens were incubated with RPA80 monoclonal antibody(diluted 1:200,Santa Cruz,USA).Immunohistochemical staining was performed using UltraSensitiveTM S-P kits(Maixin-Bio;PR China)according to the manufacturer’s instructions and using the reagent supplied with the kit.PBS was used in place of the primary antibodies for negative control.The staining intensity and area extent were scored by the German semi-quantitative scoring system as previously described8.Breast cancer cell lines and cell culture conditionsBreast cancer cell lines including MCF-7(chemotherapy responsive,ER+,PR+/-,Her-2-,Ki67 low),ZR-75(usually endocrine responsive,variable to chemotherapy,ER+,PR+/-,HER2+,Ki67 high)and MDA-MB-231(Intermediate response to chemotherapy,ER-,PR-,HER2-)were chosen for this study and maintained under recommended culture conditions.They are obtained from the American Tissue Culture Collection(Manassas,USA)and stored in the laboratory of Pathology Department,the First Affiliated Hospital and College of Basic Medical Sciences of China Medical University(Shenyang,China).All the cells were cultured in RPMI 1640 medium(Gibco,USA)or DMEM medium(Gibco,USA)supplemented with 10%fetal bovine serum(FBS)(Hyclone,USA)in a 5%CO2 humidified atmosphere at 37℃.Western blot analyses and quantitative real-time PCR(qRT-PCR)analyses of RAP80 in breast cell lines or breast tissuesThe breast tissues or cells were washed with ice-cold phosphate-buffered saline and then lysed in lysis buffer containing 10 mM Tris(pH 7.5),1%sodium dodecyl sulfate(SDS),10 mM ethylenediaminetetraacetic acid(EDTA),150 mM NaCl,1 mM sodium orthovanadate and a mixture of protease inhibitors(1 mMphenylmethylsulfonyl fluoride,1 μg/ml pepstatin A,2 μg/mL aprotinin).The lysates were sonicated for 10 s,centrifuged for 20min at 12000rpm.Equal amounts(25 μg)of the cell lysates were resolved by 12%SDS-PAGE and transferred to polyvinylidene fluoride membranes.After blocking,blots were incubated mouse anti-RAP80 monoclonal antibody(No.14466,diluted 1:400,Cellsignal,USA)or β-actin(Zhongshan Golden Bridge Biotechnology,1:1000)overnight at 4℃ and followed by each corresponding second antibody at room temperature for 1h at 37℃.Then the results developed by ECL(Pierce Biotechnology,USA).The protein bands were then analyzed using the BioImageing System(UVP,USA).The grayscale values of the RAP80 was normalized to the values of the correspondingβ-actin band to determine the expression level of the protein.The experiments were repeated at least three times independently.Total RNA from breast cell lines was extracted with Trizol reagent and the quality of RNA was analyzed by A260/A280 ratio by NanoPhotometer.The ratios were between 1.6 and 1.8.The reverse transcription was performed with RNA PCR Kit(AMV Ver.3.0,Takara,Japan)according to the manufacturer’s protocols.RT-PCRq was performed by SYBR? Premix Ex TaqTM II Kit(Takara,Japan)Using 7500 Real-Time PCR system.2μl template cDNA were added to the final volume of 20μl of reaction mixture.Expression of the selected genes were normalized to GADPH,which was used as an internal housekeeping control.The experiments were repeated at least three times independently.The results showed that mRNA and protein of RAP80 were obvious in MCF-7 and very weak in ZR-75 or MDA-MB-231.So we picked MCF-7 to be transfected with RAP80 siRNA,and further to investigate the role of RAP80 in MCF-7.RAP80 siRNA transfectionThe human RAP80 gene sequence was obtained from GeneBank.According to the design principle of siRNA,an siRNA target Designers(GenePharma,Shanghai)was used to design siRNAs and synthesized targeting the specific RAP80.The MCF-7 was cultured in a 24-well plate for 24 h before the experiment of transient transfection.The cells were transfected with Lipo fectamine(Invitrogen,Carlsbad,CA)according to the manufacturer’s instruction.Following the transfection,the cells were harvested at 48 h to measure the protein levels.Control RAP80 siRNA and negative control siRNA were used in Western blot to verify the downregulation of RAP80.Control study using non-specific siRNA was carried out under the same condition.MTT assayCell proliferation was assessed at various time points by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT)assay.Briefly,2,000 cells were seeded in each well of a 96-well plate(eight repeats)and allowed to adhere for 8h.Then,cells were further treated with different concentrations of cisplatin for 24h.Then,5 mg/ml MTT(Sigma,Germen)was added to each well and incubated for 3 h.The cells were lysed by adding 150 μl/well of dimethyl sulfoxide and read at 570 nm absorbance wavelength in microplate reader.The experiments were repeated at least three times independently.IC50 values were calculated for cisplatin from the respective sigmoidal dose-response curves.Flow cytometry apoptosis assayAnalysis of apoptosis was accessed by an AnnexinV-FITC/propidium iodide double staining kit(Genmed Bioscience,China)following the manufacturer’s protocols.In brief,each group was plated in six-wells.1μM of BVDU was treated.After 48 hours of cell transfection,we added cisplatin,the cells were cultured for 24 hours and 48 hours after treatment with drugs,and then to be harvested.The experiments were repeated at least three times independently.Western blot analyses of apoptosisThe expression of apoptosis protein of each group was analyzed by Western blot as previous described using specific antibody against Fas(sc-271759,Santa Cruz,1:100),Cleaved Caspase-3(asp175)(#9661,Cell Signal,1:1000),Bcl-2(sc-7382,Santa Cruz,1:250),Apaf-1(sc-135625,Santa Cruz,1:500),Cytochrome C(sc-13156 Santa Cruz,1:200),Bax(sc-70406,Santa Cruz,1:200)or β-actin(Zhongshan Golden Bridge Biotechnology,1:1000).The experiments were repeated at least three times independently.Matrigel Invasion AssayCell invasive ability was examined using a 24-well Transwell with 8.0-μm pore polycarbonate membrane inserts(Corning Inc.,NY,USA)according to the manufacturer’s protocol.The Matrigel(100μl/ml)was applied to the upper surface of the membranes.After the treatment of transfection for 48h and 72h,cells of each group were seeded on the upper chamber(5×104 cells/well)and incubated for 18h.Cells that had invaded the surface of the membrane were fixed with methanol and stained with hematoxylin.The cells that invaded and moved onto the lower surface of the filter membrane were counted in 10 random high power fields(400×)by an inverted microscope.The experiment was repeated 5 times and the data were shown in mean ±standard deviation(S.D.).Statistical analysisSPSS version 13.0 for Windows was used for all analyses.The Pearson’s Chi-Square test was used to analyze the relationship between RAP80 expression with clinicopathological factors in breast cancer.One-way analysis of variance(ANOVA)was performed to compare data from the densitometry analysis of MTT,FAS,Western blotting and RT-PCRq and matrigel invasion assay.Statistical significance in this study was set at p<0.05.All reported p values are two-sided.Result:Expression of RAP80 in invasive breast ductal invasive cancer tissues and paired normal breast tissues by immunohistochemistryThe results of immunhistochemistry revealed mainly nucleus staining of the RAP80 protein in the mammary epithelium of paired normal breast tissues and the breast cancer cells showed staining on the nucleus.Total positive rate of RAP80 expression was 62.3%in breast ductal invasive cancer,whereas 86.75%in its paired normal breast tissues.RAP80 expression in breast cancer(62.3%,101/162)was significant lower than that in adjacent normal breast tissues(p<0.05).The relationship between RAP80 expression and different clinicopathological factors in breast cancer is shown in Table2.RAP80 expression was not with age or histological grade(p>0.05).Its expression related with tumor size,lymph node metastasis,TNM stage,ER or PR expression,Ki67 status(p<0.05).RAP80 expression related with molecular subtype(p<0.05).Expression of RAP80 mRNA in duct invasive breast cancer by qRT-PCRThe results of qRT-PCR revealed RAP80 mRNA was significantly lower in triple-negative breast cancer than other types,which was shown in Table2.RAP80 mRNA expression correlated with ER or PR expression,lymph node metastasis,Ki67 status(p>0.05).RAP80 mRNA expression wasn’t associated with age,TNM stage,CerbB-2 status or histology grade(p>0.05).The RAP80 expression in breast cancer cell lines and RAP80 siRNA in MCF-7:The mRNA and protein of RAP80 were obvious in MCF-7 and very weak in ZR-75 or MDA-MB-231.So we picked MCF-7 to be transfected with RAP80 siRNA,and further to investigate the role of RAP80 in MCF-7 biological behavior.Using western blot and RT-PCRq analysis,RAP80 protein and mRNA expression was obvious observed in RAP80 siRNA-NC transfection group or wild-type MCF-7,very weak in RAP80 siRNA transfection group.It is indicated that the effective downregulation of RAP80 in MCF-7 by RAP80 siRNA.Effect of RAP80 siRNA transfection on cell proliferation by MTT:The survival rate of both cells decreased in a dose-dependent manner and the IC50 value for cisplatin in MCF-7 RAP80 siRNA cells was 0.83μg/ml,which was 1.69μg/ml in wild-type MCF-7.RAP80 siRNA transfection up-regulated the apoptosis of MCF-7:We monitored the level of apoptosis induction by FAS.MCF-7 transfected with RAP80 siRNA and add showed different percentage of apoptosis with the siRNA-NC transfection group or the group of MCF-7 without treatment at 48h and 72h after transfection.Apoptotic cells ratio of RAP80 siRNA transfection group was present 46.8%at 48h and 52.7%at 72h after transfection,which was higher than the siRNA-NC transfection group(13.1%at 48h after transfection,10.7%at 72h after transfection)or the group of MCF-7 without treatment(15.1%at 48h after transfection,12.9%at 72h after transfection)(p<0.05).RAP80 siRNA transfection decreased invasive ability of MCF-7:Matrigel invasion and migration assays showed that the invasive and migrating ability of the MCF-7 cell was decreased with RAP80 siRNA transfection after the treatment of transfection for 72h compared to that in MCF-7 cells without any additive or control cells with scrambled siRNA(p<0.05).Effect of RAP80 siRNA in the expression of apoptosis-related proteinWesternblot results showed that the siRNA transfection upregulated the protein expression of Caspase-3,Apaf-1,Cytochrome C and Bax(p<0.05,Figure 6),downregulated the protein expression of Bcl-2(p<0.05,Figure 6).Protein levels analyzed by Westernblot also confirmed this result.Conclusion:RAP80 expression related with ER or PR activity.Inhibition of RAP80 expression can induce apoptosis in breast cancer cells and improve chemo-sensitivity to cisplatin.Tumor cells can activate protective responses to inhibit cell cycle progression which RAP80 may related with and repair cisplatin-induced DNA damage.RAP80 related with BRCA1 effect,which can be used as an interesting target forpharmacological modulation that can increase the efficiency of cisplatin chemotherapy. |
PDF Full Text Request